The activity of many biological systems is mediated by proteolytic enzymes which cleave precursor proteins at specific amino acid residues. One major type of these enzymes, the serine proteases, are so named because the initial step in their proteolysis reactions is the attack of the hydroxyl of an active site serine on the amide carbonyl at the scissile site of the protein. This results in a tetrahedral intermediate which subsequently breaks down into an acyl enzyme and the amino terminus of the cleaved sequence. Hydrolysis of the acyl enzyme releases the carboxyl terminus and the free enzyme. A subclass of the serine proteases is composed of the trypsin-like enzymes, which cleave proteins site-specifically such that the liberated carboxyl terminus is arginine or lysine.
A great deal of research has been directed at finding mechanism-based inhibitors of serine proteases. In general, this approach has involved finding small molecules or peptides that both fit into the active site and contain a functionality able to interact with the active site serine. Irreversible inhibitors would contain a functionality that forms a covalent bond with the serine residue. Of more therapeutic interest are reversible inhibitors, which would contain a functionality that interacts with the serine residue to form a transient species that mimics the tetrahedral intermediate formed during cleavage of the natural substrate.
Several researchers have experimented with boron-containing reversible inhibitors of serine proteases. The binding of boronic acids to serine proteases most likely involves initial attack of the serine hydroxyl onto boron to form a tetrahedral boron "ate" complex. This complex can serve as a mimic of the tetrahedral intermediate formed during hydrolysis of the natural substrate, as disclosed by Zhong et. al., J. Am. Chem. Soc. 113, 9429 (1991). For example, Koehler et al. in Biochemistry 10, 2477 (1971) reports that 2-phenylethane boronic acid inhibits chymotrypsin at millimolar levels. The synthesis of boronic acid analogs of N-acyl-.alpha.-amino acids has yielded more effective inhibitors. Matteson et al. J. Am. Chem. Soc. 103, 5241 (1981) described Ac-boroPhe-OH, (R)-1-acetamido-2-phenylethane boronic acid, which inhibits chymotrypsin with a K.sub.i of 4 .mu.M. More recently, Shenvi, in U.S. Pat. No. 4,537,773 disclosed that boronic acid analogs of .alpha.-amino acids, containing a free amino group, were effective inhibitors of aminopeptldases. Shenvi, in U.S. Pat. No. 4,499,082, disclosed that peptides containing an .alpha.-aminoboronic acid with a neutral side chain were more effective inhibitors of serine proteases, exceeding inhibitors disclosed earlier by as much as three orders of magnitude in potency.
The trypsin-like protease thrombin is the final protease in both the intrinsic and extrinsic pathways of the blood coagulation cascade and thus is of crucial importance in the blood coagulation process. Thrombin is responsible for the cleavage of fibrinogen to fibrin, which is then cross-linked by factor XIIIa, thereby stabilizing a developing blood clot. In addition, thrombin activates platelets and also factors V and VIII, which potentiate its own production, as described in Hemker and Beguin, Jolles et. al. "Biology and Pathology of Platelet Vessel Wall Interactions," 1986 , pp. 219-226; Crawford and Scrutton in: Bloom and Thomas "Haemostasis and Thrombosis," 1987 pp. 47-77. Inhibitors of thrombin are expected to be effective in the treatment of thrombosis, a condition in which unbalanced activity of the hemostatic mechanism leads to intravascular thrombus formation. Direct thrombin inhibitors are also expected to be devoid of the side effects of bleeding and high interpatient variability which are common with heparin and vitamin K antagonist therapy (Green et al. Thromb. res. 1985, 37, 145-153).
Several mechanism-based thrombin inhibitors have been disclosed, most notably those based on the D-Phe-Pro-Arg sequence. The chloromethyl ketone Ac-D-Phe-Pro-ArgCH.sub.2 Cl disclosed by Kettner and Shaw Thromb. Res. 14, 969 (1979) was found to be a potent and selective irreversible inhibitor of human thrombin. The corresponding aldehyde Ac-D-Phe-Pro-Arg-H, desclosed by Bajuez et. al. Folia Haematol. 109, s. 16 (1982) was shown to be a reversible inhibitor of thrombin with a Ki=75 nM. This class of reversible thrombin inhibitors is also exemplified by the trifluoromethyl ketone D-Phe-Pro-ArgCF.sub.3 disclosed by Kolb et. al., AU-B-52881/86.
This series of tripeptide thrombin inhibitors was expanded to include the boronic acid derivatives which are exemplified by Ac-D-Phe-Pro-boroArgOH in Kettner and Shenvi, European Patent Application EP 293 881. This compound has a K.sub.i =0.041 nM and is highly effective in the inhibition of blood coagulation both in vitro and in vivo. Additional boronic acid inhibitors of thrombin have been disclosed: Elgendy et al., Tetrahedron Lett. 33, 4209 (1992) have described peptides containing .alpha.-aminoboronic acids with aliphatic neutral sidechains which are thrombin inhibitors.
There have been patent disclosures which describe alternatives to the D-Phe residue in the D-Phe-Pro-Arg amino acid sequence. Metternich, in European Patent Application EP 471 651, has described peptides containing boroArginine and boroLysine which contain at least one unnatural amino acid residue. Kakkar in PCT Application WO 92/07869 has claimed peptide thrombin inhibitors of the general structure, X-Aa.sub.1 -Aa.sub.2 -NH--CH(Y)-Z where Aa.sub.1 and Aa.sub.2 are unnatural amino acid residues, Z can be a variety of electrophilic groups including boronic acid, and Y can be a variety of basic sidechains. Tripeptide agents limited to .alpha.-alkyl and .alpha.-aryl or heteroaryl substituted glycines conjugated to -Pro-Arg-H have been disclosed by Lilly in European Patent Application EP 0 479 489 A2. Sandoz has disclosed in European Patent Application EP 471 651 A2 boroLysine and boroArginine peptide analogs containing at least one unnatural hydrophobic .alpha.-amino acid substituted with groups such as trimethylsilyl or naphthyl. Balasubramanian et. al., in J. Med. Chem. 36, 300 (1993), has reported replacements for the D-Phe of D-Phe-Pro-Arg-H and found the dihydrocinnamoyl group to be effective, although somewhat less potent.
Despite the foregoing, more efficacious and specific thrombin inhibitors are needed as potentially useful therapeutic agents for the treatment of thrombosis. The present invention relates to an extensive study of non-amino acid replacements for the D-Phe of the boropeptide D-Phe-Pro-boroArgOH.